Lab 11 summary CC-BY-NC

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Summary of Lab 11 for BIOL 112 (Winter 2011). Week of Mar 21 2011.

DNA electrophoresis.

Genetics: Double recombination and mapping problems.

1DNA electrophoresis

  • To separate DNA fragments based on their size
  • Electrophoresing them through agarose gel
  • In this lab: Genomic DNA, plus DNA fragment containing the fruit fly gene cdk cloned into a plasmid
    • Genomic DNA more complex than the cloned cdk gene
  • Procedure:
    • Comb template placed in tray
    • Agarose gel heated, cooled, poured into tray; solidifies; comb template removed
    • To load DNA samples, use pipetter, try to place it inside well (kind of tricky)
    • DNA samples contain bromophenol blue - blue dye, to monitor progress of electrophoresis
    • Once the samples have been loaded, close, connect to power supply for a while
  • What is cloning genes:
    • Purifying a particular gene away from the rest of the genomic DNA, putting it in a bacterium
    • To make the bacterium accept the carrier gene, we put it in carrier DNA called plasmids
    • We also give it antibiotics to give the bacteria a reason to accept the plasmids lol
    • To do this, we cut the plasmid open with a restriction enzyme
    • Restrictions enzyme cut any DNA at a specific sequence, usually 6 base pairs, called a restriction site
    • Then we cut the genomic DNA with the same enzyme, so hopefully the two end pieces stick together, mixing genomic and plasmid DNA
    • Then, put the plasmid+cdk DNA back into bacteria (process: transformation)
    • We throw in an an antibiotic, and only the bacteria that have accepted the plasmid DNA will grow (for the most part)
    • To make sure we only get bacteria with the DNA we want
    • One problem - how do we make sure we have the right DNA attached to the plasmid etc
      • Look at its restriction map using a restriction digest
      • Cut the gene with restriction enzymes, see if it has the correct restriction sites in correct places etc
      • Whatever
  • Large fragments of DNA migrate more slowly than smaller fragments (so less distance travelled)

2Double recombination

  • If you have three pairs of genes, all on the same chromosome
  • Double recombination should occur less frequently than single recombination (but less frequently than expected, due to interference)
  • First, determine the order of the genes - abc or bac or bca
  • Then identify the double recombinants - the least frequent class of progeny
    • Use that to figure out which gene is in the middle
    • Distance between middle gene and one outer gene: frequency of just that outer gene switching (%)
    • Same for other outer gene
    • Distance between outer genes: add the two distances above